App Papp Eng

Health Products and Food
Branch Inspectorate
Holland Cross, Tower "A"
2nd Floor, 11 Holland Avenue
Address Locator # 3002C
OTTAWA, Ontario
K1A 0K9
May 30, 2003
03-100069-318
TO: ASSOCIATIONS
I am pleased to inform you that a revised version of Guide 0006

entitled "Process validation: Aseptic Processes for Pharmaceuticals" is
now available on the Health Products and Food Branch Inspectorate website
at the following address:
www.hc-sc.gc.ca/hpfb-dgpsa/inspectorate
This document has been revised to harmonize its content with version
2 of the Good Manufacturing Practices (GMP) Guidelines, 2002 Edition. No
major changes to the requirements were made, therefore, consultation is
not deemed necessary.
Inquiries about this document can be addressed to Ms. France

Dansereau, Manager, Inspection Unit, at the National Coordination Centre,
by telephone at (613) 957-1492, by fax at (613) 952-9805 or by e-mail at
france_dansereau@hc-sc.gc.ca.
Original signed by
Danièle Dionne (for)
Jean Lambert
Director General
Health Canada
Health Products and Food Branch
Health Products and Food Branch Inspectorate
GUIDE
Process Validation :
Aseptic Processes for Pharmaceuticals
Supersedes:
May 1, 2001
Date issued:
April 1st, 2003
Date of implementation:
June 1st, 2003
Ce document est aussi disponible en français.
1
TABLE OF CONTENTS
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Validation - General/Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3. Protocol Development and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4. Personnel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5. Data Review and Study Certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
6. Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
7. Environmental Considerations: Clean Room Standards, Qualification and Monitoring . . 9
8. Equipment Qualification and Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
9. Media Fill Studies (Solution Products) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
10. Media Fill Studies (Non-Solution Products) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
11. Revalidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
12. Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
GMP Committee members . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2
Process Validation : Aseptic Processes for Pharmaceuticals
1. INTRODUCTION
This document is intended to provide pharmaceutical dosage form manufacturers with guidance on
the validation of aseptic manufacturing processes, as required in Division 2, Part C (Good
Manufacturing Practices) of the Food and Drug Regulations, and in a manner which is acceptable
to the Health Products and Food Branch Inspectorate.
Sterile Products may be broadly classified into two main categories, according to the manner in
which they are produced: those which are sterilized after the product has been filled and sealed in
the final container(s) ("terminally sterilized" products) and those where the sterilization stage (or
stages) takes place before the bulk product is filled. In this latter instance, all subsequent processing
(typically, the filling and sealing operations) must be conducted aseptically in order to prevent
recontamination of the sterilized product.
It is recognized that aseptic processes play an important role in rendering sterile formulations which
cannot be terminally sterilized. However, terminal sterilization, in particular using moist heat
processes, is considered to be the method of choice in the manufacture of sterile products due to the
enhanced sterility assurance which it affords. Manufacturers who choose to manufacture a sterile
product without terminal sterilization must be prepared to justify this decision by demonstrating that
the product cannot be terminally sterilized, even under less severe autoclave cycles tailored to the
bioburden of the batch (Probability of Survival approach).
The two most common pharmaceutical applications of aseptic processing methods are (a) the filling
of liquid products following sterilization by filtration and (b) the filling of previously sterilized bulk
powder products. Both are covered in this guide. The final section of this guide outlines
documentation required to provide acceptable evidence that a given process has been thoroughly
evaluated and is adequately controlled.
It is assumed that, throughout, manufacturing and control operations are conducted in accordance
with the principles of Good Manufacturing Practice, both in general and in specific reference to
Sterile Products manufacture.
The steps recommended in this guideline may be summarized as follows:
S As a pre-requisite, all studies should be conducted in accordance with a detailed, pre-

established PROTOCOL, or series of protocols, which in turn is subject to formal
change-control procedures.
(See Section 3).
S Both the personnel conducting the studies, and those running the process being
studied should be appropriately TRAINED and QUALIFIED and be suitable and
competent to perform the tasks assigned to them (See Section 4).
S All data generated during the course of the studies should be formally REVIEWED
and CERTIFIED, as evaluated against pre-determined criteria (See Section 5).
May 2003 3
- Suitable TESTING FACILITIES, EQUIPMENT, INSTRUMENTS and
METHODOLOGY should be available (See Section 6).
S Suitable CLEAN ROOM FACILITIES should be available, in terms both of the

"local" and "background" environments. Assurance that the Clean Room
environment is as specified should be secured through initial commissioning
("Qualification") and subsequently through the implementation of a program of re-
testing, in-process control and monitoring (See Section 7).
S All processing equipment should be properly INSTALLED, QUALIFIED and

MAINTAINED (See Section 8).
S When appropriate attention has been paid to the above, the aseptic process may be
validated by means of "MEDIA FILL", (or "PROCESS SIMULATION") studies
(See Sections 9 and 10).
S The process should be REVALIDATED at intervals (See Section 11).
S Comprehensive DOCUMENTATION should be available to define, support and

record the overall validation process (See Section 12).
Whilst this Guide is concerned only with the validation of ASEPTIC PROCESSES, it is crucial to
the success of any such process that the product, materials, components etc. that are being
handled/processed aseptically (e.g. bulk solution or powder; containers and closures) plus any
equipment, vessels or surfaces (e.g. holding tanks, pipework, filling machines) which will or can
come into contact with sterilized products/materials have themselves been previously sterilized by
appropriate and validated sterilization processes. In any aseptic filling process, assurance of
container/closure integrity is, of course, vital. Evidence that all this is so should be provided as part
of the overall Validation Documentation (see Section 12).
2. VALIDATION - GENERAL/TERMINOLOGY
2.1 In the context of this guide, Process Validation is defined as:
The action taken to demonstrate, and to provide documented evidence that

a process will, with a high degree of assurance, consistently achieve the
desired and intended results.
2.2 Before Process Validation can commence there must be what may be termed an
essential Prevalidation phase. This phase, in addition to such considerations as
equipment specification, equipment design and equipment purchase, requires
attention to Equipment Qualification.
2.3 Equipment Qualification in turn has two main phases :
2.3.1 Installation Qualification, that is demonstrating and certifying that a

piece of equipment is properly installed, is provided with all
May 2003 4
necessary services, subsidiary equipment and instruments, and is
capable of performing in accordance with its basic design parameters.
2.3.2 Operational Qualification, consists of demonstrating that the

equipment will perform consistently, and within pre-defined limits,
as specified and installed.
2.4 None of these various phases need to be considered as entirely "water-tight"

compartments. The divisions have been defined as a matter of convenience in
discussion. In practice there is likely to be some overlap, or merging, between the
various components of Validation/Qualification. In addition, there are quite wide-
spread variations in terminology and conception. Some consider "Qualification" and
"Validation" as two separate, yet related activities. Others use the term "Validation"
to embrace the overall activity of Prevalidation/Qualification PLUS Process
Validation.
The relationships between these various phases may be summarized as
follows:
Specification Equipment Qualification Process

Design Qualification
Purchase Installation Operational
Qualification Qualification
Sometimes termed “Prevalidation”
The overall activity of Validation
2.5 Validation has also been considered to have three aspects, or possible
strategies - Prospective Validation, Concurrent Validation, and
Retrospective Validation.
2.5.1 Prospective Validation applies to new processes and new equipment,

where studies are conducted and evaluated, and the overall
process/equipment system is confirmed as validated before the
commencement of routine production.
2.5.2 Concurrent Validation applies to existing processes and equipment.

It consists of studies conducted during normal routine production and
can only be considered acceptable for processes which have a
manufacturing and test history indicating consistent quality
production.
2.5.3 Retrospective Validation applies to existing processes and equipment,

and is based solely on historical information. Unless sufficiently
detailed past processing and control records are available,
retrospective validation studies are unlikely to be either possible or
acceptable. For example, it would be
May 2003 5
necessary to establish that the process had not been modified and that
the equipment was still operating under the same conditions of
construction and performance as documented in the historical
records. Maintenance records and process change control
documentation would be necessary to support any such claim.
Furthermore, the incidence of process failures, and records of rejects
and/or reworking would need to be carefully evaluated for evidence
of inconsistency in the process. Manufacturing, maintenance, testing
and calibration data would all need to demonstrate process
uniformity, consistency and continuity.
2.5.4 Concluding Note on Validation Terminology. While there is
considerable variation in the understanding and use of the various
terms discussed above, there is general agreement that the critical
validation concepts are the following:
S the overall process is understood

S equipment is appropriately specified and designed
S equipment is properly installed and maintained and is
demonstrably operating as specified and designed
S the process is validated to ensure that it does achieve the
desired and intended result.
3. PROTOCOL DEVELOPMENT AND CONTROL
3.1 Each stage in the validation of the overall process should proceed in accordance with
a pre-established and formally approved, detailed, written protocol, or series of
related protocols.
3.2 Prior to the commencement of the studies, written change control procedures should
be established, which will prevent unauthorized changes to either the process itself,
or to the study protocol, and restrict change during any stage of the study until all
relevant data are evaluated.
3.3 Protocols should have a Title, Date and a unique Identification or Reference Number.
They should be formally authorized/approved by person(s) with the competence and
authority to do so.
3.4 Protocols should specify the following in detail:
3.4.1 The objectives and scope of the study. That is, there should be a clear
Definition of Purpose.
3.4.2 A clear and precise definition of the process, equipment, system or sub-
system which is to be the subject of the study, with details of performance
characteristics.
3.4.3 Installation and qualification requirements for new equipment.
May 2003 6
3.4.4 Any up-grading requirements for existing equipment, with justification for
the change(s) and a statement of qualification requirements.
3.4.5 Detailed, step-wise statement of actions to be taken in performing the

study (or studies).
3.4.6 Assignment of responsibility for performing the study.
3.4.7 Statements on all test methodology to be employed, with a precise statement

of the test equipment and/or materials to be used.
3.4.8 Test equipment calibration requirements.
3.4.9 References to any relevant Standard Operating Procedures (SOPs).
3.4.10 Requirements for the content and format of the report on the study.
3.4.11 Acceptance criteria against which the success (or otherwise) of the study is
to be evaluated.
3.4.12 The personnel responsible for evaluating and certifying as acceptable each
stage in the study, and for the final evaluation and certification of the process
as a whole, all as measured against the pre-defined acceptance criteria.
4. PERSONNEL
As with all Process Validation studies, documented evidence of the relevant experience and training
of the personnel involved in conducting the studies should be maintained. However, because the
personnel actually performing the aseptic processing (both during the course of any validation
studies, and in routine operation) can, and do, have so crucial an effect on the quality of the end-
product, it is appropriate and necessary to consider both these aspects of personnel involvement.
4.1 Appropriately qualified personnel should ensure that the protocol and the testing
methodology are based on sound scientific principles and that all studies are properly
evaluated and certified.
4.2 All personnel conducting tests should be trained and experienced in the use of the
instruments, measuring devices and materials used.
4.3 Engineering/maintenance personnel should be fully trained and competent in the

operation and maintenance of the machines, equipment, and air control systems
involved.
4.4 Although modern automated and barrier techniques may reduce contamination risk,
the significance of the "human factor" in all aseptic processing operations cannot be
over-stressed. For the results of any validation studies themselves to be valid, it is
May 2003 7
essential that the risk represented by so potentially random a variable as a human
operator is kept as much under control as is possible. That is, steps must be taken
to reduce the risk and to minimize the variability.
4.5 This in turn means that any operators involved in performing an aseptic processing
operation which is the subject of a validation study should adopt the same
techniques, disciplines, and standards of hygiene, clothing and behaviour as in
normal routine manufacture. The converse also applies: if operators conduct
themselves, during routine production, in manner which is different from their
behaviour etc. during the validation studies, then conclusions drawn from the
validation will be invalid.
4.6 It is therefore vital that all personnel involved in aseptic processing operations are
trained in, and fully understand, the concepts and principles of GMP, and the
relevant elements of microbiology. They must understand the importance of
personal hygiene and cleanliness, and be made fully aware of the possible hazardous
consequences of product contamination.
4.7 Operators should be provided with suitable Clean Room clothing and trained in
appropriate gowning technique. The type of clothing to be worn, and the "scrub-up"
and gowning process should be defined in written procedures, available to the
operators, and preferably displayed in the changing room. The same
clothing/gowning standards should be observed during validation studies as in
routine production, and vice versa.
4.8 The maximum number of personnel permitted in the Clean Room during normal
routine production should also be present in the Clean Room during any validation
test runs.
4.9 At all times, operators should be encouraged to report any infections, open lesions
or any other conditions which could result in the shedding of abnormal numbers of
particles or microorganisms. As with routine manufacture, no person thus affected
should be present in the Clean Room during validation test runs.
4.10 As in routine production, Clean Room operators involved in validation studies

should be microbiologically monitored by taking test samples from gloves, gowns
and facemasks.
4.11 Normal routine process documentation should specify and record the numbers and
types of operator interventions that are permitted during processing, and in what
circumstances. A similar series of interventions should occur during any validation
test runs. Details should be provided as part of the overall validation documentation
(See Section 12).
Note: As stated in the Introduction it is assumed that all routine manufacturing and control
operations are conducted in accordance with Good Manufacturing Practice, and this
includes a requirement that all personnel are trained and competent to carry-out the
tasks assigned to them.
May 2003 8
5. DATA REVIEW AND STUDY CERTIFICATION
5.1 All information or data generated as a result of the study protocol should be
evaluated by qualified individuals against protocol criteria and judged as meeting or
failing the requirements. Written evidence supporting the evaluation and
conclusions should be available.
5.1.1 These evaluations should be made as the information becomes available.
5.1.2 If such an evaluation shows that protocol criteria have not been met, the
study should be considered as having failed to demonstrate acceptability, and
the reasons should be investigated and documented.
5.1.3 Any failure to follow the procedure as laid down in the protocol must be
considered as potentially compromising the validity of the study itself, and
requires critical evaluation of the impact on the study.
5.1.4 The final certification of the validation study should specify the pre-
determined acceptance criteria, against which success or failure was
evaluated.
6. LABORATORY
6.1 All laboratory tests (including physical, chemical and microbiological

determinations) should be performed by a competent laboratory, suitably equipped,
and staffed with personnel properly trained and qualified to carry-out the test
procedures assigned to them.
6.2 Detailed authorized, written procedures defining the relevant, validated methodology
should be available for all laboratory tests which are to be carried out during the
course of the study. These procedures should be referenced in the study protocol.
6.3 If any external laboratory facilities are used, a system should be in place for
determining the competence of these laboratories to carry out the tests required. This
requirement should be referenced in the study protocol.
6.4 All measuring/recording/indicating instruments employed in the studies should be

adequate for the purpose, in terms of range, accuracy, reproducibility, etc... They
must be calibrated in accordance with pre-defined written procedures before any
validation studies are commenced.
6.5 Records of each calibration should be maintained, and should form part of the overall
validation documentation.
May 2003 9
6.6 For the conclusions drawn from any qualification/validation studies themselves to
remain valid during routine production, all controlling and recording instruments
must be subjected to a written maintenance and calibration program.
7. ENVIRONMENTAL CONSIDERATIONS: CLEAN ROOM STANDARDS,

QUALIFICATION AND MONITORING
7.1 Although prior to their being sterilized, products, materials, containers, components,
closures etc. may be handled/processed in a lower (for example, Grade C) Clean
Room environment, subsequent to the sterilization stage(s) all aseptic processing
operations should be conducted under local Grade A ("work station") protection,
within a general (or "background") Grade B Clean Room environment. However,
if certain specialized automated or barrier techniques are employed to provide the
localized protection, a lower background environmental standard may be acceptable,
provided that process validation studies demonstrate the attainment of an acceptable
level of sterility assurance. (Grades A, B and C are as defined in the table for the
“Basic Environmental Standards for the Manufacture of Sterile Products” in the
Sterile Products section of the current version of the "Good Manufacturing
Practices".
7.2 For the results of any validation studies to have valid relevance to routine production,
they must be conducted under precisely the same environmental conditions as used,
or intended to be used, during normal routine production.
7.3 Confirmation and Certification that the room and the work station(s) do, in fact
conform to the specified Environmental Standard may be considered as forming part
of the Installation Qualification phase. To this end, the following basic work should
be carried-out on the initial commissioning (or "Qualification") of a new Clean
Room installation:
S Room air filter integrity tests.

S Determination of air velocity at the face of each air inlet
filter.
S Room air change rate.
S Room air particle counts.
S Room air pressure differentials and air flow patterns.
S Lighting, heating, humidity.
S Work station(s) air filter efficiency tests.
S Determination of air velocity at face of work station air
filters.
S Particle counts within work station areas.
7.4 Following the initial commissioning, a regular re-test program should be adopted,
e.g.:
May 2003 10
7.4.1 Room and Work Station Air Filter Tests: Repeat at least annually, unless
results of normal in-process monitoring indicates a need for more frequent,
or additional testing.
7.4.2 Air Velocity and Room Air Changes: Repeat at least twice a year.
7.4.3 Air Particle Counts: Determine as part of regular in-process monitoring, with
formal certification by a competent specialist agency 3 times per year.
7.5 Room pressure differentials should be monitored on a continuous, on-going,

basis.
7.6 Walls, floors, work stations and surfaces generally should be subject to a pre-
determined program of cleaning and disinfection.
7.7 In order to ensure that, during routine manufacture, products remain within the
quality parameters established during the overall validation process, it is necessary
to design and implement a program of in-process control and monitoring. Similarly,
as part of the over-all assurance that process validation studies are conducted under
comparably normal processing conditions, a similar in-process control and
monitoring program should be operated during the process validation runs.
7.8 In-process monitoring and control may be considered under three headings:
S Environmental Particulate
S Microbiological
S Filter Integrity Testing
7.9 As appropriate to the type of manufacturing process, consideration needs to be given

to the following Microbiological Monitoring and Control Procedures:
S Bioburden check on bulk solution, prior to sterile filtration.
S Exposure of "Settle Plates" (Petri dishes of nutrient agar) at

critical positions within the general Clean Room environment
and at the controlled work station(s).
S Use of Air Sampling devices to determine the number of

viable organisms per cubic metre (or cubic foot) of air in the
room, and within the work station(s).
S Use of Contact Plates, or Swabs, to check the microbiological

quality of surfaces.
7.10 Environmental Particulate monitoring should be carried out using appropriate air
Particle Counting devices to check that the general environmental and work station
air remain in conformity with specification.
May 2003 11
7.11 Filter integrity testing of the filter(s) used to sterilize the product is critical in sterile
product manufacturing. If the product cannot be sterilized in the final container,
solutions or liquids can be filtered through a sterile filter of normal pore size of 0.22
micron (or less), into a previously sterilized container. The integrity of the sterilized
filter should be verified before use and should be confirmed immediately after use
by an appropriate method, such as a bubble point, diffusion, or pressure hold tests.
7.12 This in-process monitoring and control should be conducted in accordance with a
written, pre-determined program, which includes specified test limits and standards,
and with all results formally reported and evaluated against those limits. This
requirement applies as much to validation studies as routine manufacture.
8. EQUIPMENT QUALIFICATION AND MAINTENANCE
8.1 A wide range of different types of mechanized equipment may be used in various
aseptic processing operations. Before any process validation studies may be
commenced, it is necessary that all such equipment be properly qualified, in both
Installation and Operational terms (see 2.2 and seq.), and that this qualification be
certified. It is clearly outside the scope of these guidelines to detail Installation and
Operational requirements for every possible item of equipment. The essential
requirements are that the equipment be:
S Confirmed as having been constructed as specified.
S Properly installed and provided with all necessary functioning

services, ancillary equipment and instruments.
S Confirmed as capable of operating consistently, within pre-

determined limits, over its defined operating range.
8.2 Processing equipment must be confirmed as Qualified before any subsequent studies
can be considered valid.
8.3 For the results of any validation studies themselves to remain valid in routine
manufacture, a comprehensive routine maintenance program should be developed,
setting out each activity in detail along with the frequency in terms of real time,
machine time or other time base. The time base should be clearly defined for each
procedure.
8.4 Unless such a program is developed and implemented, and the manufacturing
equipment and attendant instruments remain in the same state as during the
validation studies, then any assurance derived from those studies could be considered
to be negated.
9. MEDIA FILL STUDIES (SOLUTION PRODUCTS)
May 2003 12
9.1 The "Media Fill", or "Broth Fill", technique, is one in which a liquid microbiological
nutrient growth medium is prepared and filled in a simulation of a normal
manufacturing operation. The nutrient medium processed and handled in a manner
which simulates the "normal" manufacturing process as closely as possible with the
same exposure to possible contamination (from operators, environment, equipment,
and surfaces) as would occur during routine manufacture. The sealed containers of
medium thus produced are then incubated under prescribed conditions and examined
for evidence of microbial growth, and thus of an indication of the level of
contaminated units produced. The process is summarized in Figure 1
May 2003 13
Figure 1: Process Flow Diagram of Liquid Media Filling of vials
Vials Stoppers Overseals Dispense Medium Powder
Wash Wash Wash Prepare Solution
Sterilize Sterilize Sterilize Sterilize Liquid Medium
Fill / Stopper / Seal Aseptically
Inspect
Inoculate Control Vials
Incubate
"Read" Results
NOTES:
S Different types of containers will require different methods of sterilization. For
example glass vials are likely to be dry heat sterilized, plastic vials may be sterilized
by irradiation or ethylene oxide.
S Any other components, e.g. teats/droppers will also need to be pre-sterilized by some
suitable validated method.
May 2003 14
S The process flow for liquid media filling of ampoules will be analogous to the above,
without the operations involving stoppers, overseals etc...
9.2 It is important to recognize that, in many instances, media fills are, amongst other
things, a test of the human operators' aseptic techniques. In this test situation these
operators can hardly remain unaware that nutrient medium is being filled, and that
they themselves are, to an extent,"under test". There is, therefore the possibility that
they will take more than their usual care, and thus the normal process will not be
precisely simulated. Every effort should be made to ensure that the operators do
behave normally during the media fills, and conversely (and perhaps importantly)
that during routine production they do not deviate in any way from the high
standards adopted during those simulation studies.
9.3 A further difficulty which needs to be noted is the possibility of contamination of the
facility and equipment by the nutrient medium. If the process is well controlled and
the media-fill is promptly followed by cleaning and disinfection, and (as necessary)
sterilization of equipment, contamination should not occur. Nevertheless, it is
important to recognize the potential hazard, and to respond accordingly.
9.4 It must also be emphasized that the filling of a nutrient medium solution alone does
not constitute an acceptable aseptic process validation. The whole manufacturing
cycle must be simulated, from the dispensing and reconstitution of the powdered
medium under normal manufacturing conditions, to the filling and sealing process
itself. Operators (and numbers of operators), numbers and types of filtrations etc.
should all be "as normal", as should holding times in any mixing vessels, interim
holding tanks etc. General activity should be at a normal level, and no attempt
should be made to take any "special" precautions to ensure that the test run is
successful. If any deviation from the normal is permitted, it should only be in the
direction of presenting a greater, rather than a lesser, microbiological challenge to
the process.
9.5 Before any meaningful aseptic process validating media-fills can be carried-out, all
necessary Equipment Qualification and Instrument Calibration must be completed,
together with the appropriate certification (see e.g. Sections 6 and 8). The Clean
Rooms used for all processing stages should also have been confirmed and certified
as complying with the required environmental standards. (See Section 8).
9.6 Normal routine in-process control and monitoring procedures (see Section 8) should
be operated during the media-fills.
9.7 The liquid Nutrient Medium used should meet the following criteria:
Selectivity: The medium should have low selectivity, that is, it should be
capable of supporting growth of the widest range of micro-
organisms that might reasonably be encountered.
May 2003 15
Clarity: As "made-up", it should be clear, so as to allow for

the observation of any evidence of growth following
incubation.
Filterability: Where the process being simulated includes a filtration stage,

the liquid medium should be capable of being filtered through
the same grade and type of microbial retentive filter as that
through which the actual product is, or will be, filtered.
Liquid Soybean Casein Digest (SCD), also termed "Tryptic
Soy Broth" (TSB) is perhaps the liquid medium most
frequently employed. However, other formulations (for
example, liquid Tryptone Glucose Yeast Extract, Brain Heart
Infusion etc.) may be used, provided they meet the criteria set
out above.
9.8 The liquid medium should be either sterilized by filtration (if such a stage is
part of the normal operation being simulated) or pre-sterilized by heat and
cooled to ambient temperature before proceeding.
9.9 The Number of Units to be filled per run should be sufficient to provide a
high probability of detecting a low incidence of microbial contamination.
For example, in order to give 95% confidence of detecting a contamination
rate of 1 in a thousand units filled (i..e. 0.1%) with sterile nutrient media,
3000 units need to be filled and no contaminated unit should be found after
the incubation period. (However, see 9.19).
9.10 For the initial validation of a new process or facility, sufficient consecutive media
fill runs should be performed to provide assurance that the results obtained are
consistent, meaningful and provide an acceptable level of sterility assurance. At
least 3 separate, consecutive, successful runs per operator team, or shift, should be
performed to acceptable initial validation of a given process line (For Revalidation,
see Section 11).
9.11 The Volume to be Filled per unit should be the normal production fill-volume where
possible. In the case of high volume containers, a lesser quantity may be used,
provided steps are taken to ensure wetting of all the inner surface of the container,
and any closure, by the medium, e.g. by shaking or inversion, and/or by inverting the
containers part-way through the incubation period. It is good practice to take similar
steps to ensure complete inner surface wetting when normal full volumes are filled
as well.
9.12 Immediately following filling, all units filled should be examined for leakers and/or
damage. In this context, any leak-test method in which heat is employed should
obviously not be used. Any leakers or damaged units should be rejected.
9.13 Incubation of the filled units should follow immediately after filling and leak-testing,
and should be for a period of 14 days.
May 2003 16
9.14 The Incubation Temperature should be 30oC to 35oC. Incubation temperatures

should be carefully monitored and maintained throughout the incubation period.
9.15 Test Controls: Media used in the evaluation must pass a growth promotion test where
a challenge with between 10 - 100 organisms per container is suitable to show the
growth characteristics of the organism.
9.16 Reading of Results: All units filled and incubated should be visually examined for
microbial growth after 14 days incubation. Any contaminated units will be
identifiable by the turbidity of the medium. Any contaminated units that are found
should be examined in the laboratory, and the contaminating organisms identified,
to the species level where possible, so that appropriate preventative action may be
taken. For the results of the media fill run to be considered valid, all the inoculated
control units should display growth.
9.17 The contamination rate found in a media fill run should be calculated as follows:
Observed number of 0 1 2 3 4 5 6 7 8 9 10
failures
Upper 95% confidence 3.0 4.74 6.3 7.75 9.15 10.51 11.84 13.15 14.43 15.71 16.96
limit
Contamination Rate = Upper 95 % contamination limit x 100

Number of filled units
9.18 Acceptance Criteria: A currently accepted limit is 0.1% at a 95% confidence level.
9.19 It is however important to recognize that, for example, a media fill run of 3000 units
will usually represent only a simulated sample of a normal production run. Actual
production runs are likely to be much larger. The contamination level determined
from a media fill will therefore be subject to sampling error, such that (for example)
3 contaminated units in a media fill of 3000 may be indicative of a potential
contamination rate in actual production significantly greater than 0.1%.
9.20 The following table indicates the maximum permitted number of contaminated units
per various Media-Fill “run sizes” to indicate a 0.1% contamination limit with a
95% Confidence Level.
Media Fill Units Contaminated Units Permitted

3 000 0
4 750 1
6 300 2
7 750 3
9 150 4
May 2003 17
10510 5
11840 6
13150 7
14430 8
15710 9
16960 10
Thus for example, to provide confidence (95%) of complying with the 0.1% limit,
4750 media-filled units would be required with no more than one unit found
contaminated, or 6,300 units with no more than 2, and so on.
9.21 If batches smaller than 3,000 units are produced, the minimum number of containers
used for process simulation with sterile nutrient media should be equal to the
commercial batch size and no contaminated unit should be found after the incubation
period.
9.22 To demonstrate compliance with a contamination limit of one in 10,000 (0.01%)

notably larger numbers of units would need to be filled with "broth". For example,
in relation to a normal production run of 50,000 units, over 46,000 units would need
to be filled with medium, with no more than one unit found contaminated.
9.23 These statistical considerations reveal a distinct practical problem with regard to the
number of units which may need to be filled with medium and incubated, particularly
in any attempt to demonstrate a probability of a low (for example, less than 0.1%)
level of contamination in "standard" production batch sizes. Manufacturers should
determine (according to their particular circumstances and production batch sizes)
media-fill run sizes, with permitted contamination levels, which will provide
adequate confidence in sterility of actual production batches. Purely on the basis of
the practical limitations of the test procedure itself, a contamination level in a media
fill of 0.1%, detected infrequently, may be considered to be acceptable. Regular, or
common, contamination levels (in media fills) of 0.1% or above should be regarded
as unsatisfactory.
9.24 Whilst it may be statistically unsound to sum in a simple fashion data from a series
of discrete events, and then treat these data as if they had been derived from a single
event, a series of "good" media fill results over a period of time (assuming
reasonable comparability of conditions etc.) may be regarded as confidence-
strengthening, if not in any precisely quantifiable fashion.
10. MEDIA FILL STUDIES (NON-SOLUTION PRODUCTS)
May 2003 18
The same general principles, conditions and statistical considerations as set-out in Section 9 apply,
but the various types of non-solution Sterile Products require various adaptations to the approaches
already described. In all procedures involving the use of growth media it is vital to control any
contamination by the media of equipment, surfaces etc. All media fill studies should be promptly
followed by application of thorough cleaning, disinfecting and sterilization procedures.
10.1 Sterile Powders
The use of the media fill technique in the validation of the filling of sterile
powder products presents certain special problems, arising from the probable
necessity to employ additional equipment, techniques or manipulations which
are different (or additional) to those used in routine production. In such
circumstances the media-fill cannot unequivocally be said to be a precise
process simulation. This inevitable shortcoming may, however, have to be
accepted. A number of different approaches have been proposed and used,
as follows:
10.1.1 The normal process is simulated as closely as possible, but instead of

filling a powder, a sterile liquid medium is filled. This approach is
virtually the same as that described for a solution product (Section 9
above) and fails to simulate the actual powder fill.
10.1.2 The normal process is simulated as closely as possible, with a sterile,

dry inert powder filled in place of the normal product or material.
Lactose, mannitol and polyethylene glycol 8000 are examples of
"simulation" powders which have been used. There are two possible
variations on this approach:
a) Fill the chosen inert powder into the containers (e.g.

ampoules/vials) which are already filled with sterile
liquid medium.
b) Fill the inert powder first, and then add the sterile
liquid medium. In both these variations, a powder fill
is simulated, but an additional, non-routine step (i.e.
the filling of the liquid growth medium) is involved.
10.1.3 Fill sterile dry powdered medium into the containers, in simulation of the
normal powder filling operation, aseptically adding sterile aqueous diluent
on-line, to form liquid medium solution. As in 9.1.2, a powder fill is
simulated, but an additional operation is involved.
10.2 Whichever approach is adopted, it is important to ensure that any

powder/medium/diluent combination used does not cause growth inhibition through
hyperosmolar or other antimicrobial effects.
10.3 Suspension Products: Simulate the entire normal process as closely as possible, using
a sterile inert powder in place of the normal powder product. Micronize etc. (if this
May 2003 19
is part of the normal process) and form suspension, using sterile liquid growth
medium in place of the normal liquid phase of the suspension product. Fill as normal
and incubate. (Comments as in 10.2 above similarly apply.)
10.4 Freeze-dried Product: Simulate the entire normal process (i.e. preparation of bulk
solution, filling of solution, loading of freeze-dryer, running of freeze-drying cycle,
sealing/closing of containers, inspection) but using a liquid growth medium
(dispensed as a powder, dissolved and sterilized) in place of normal product. Actual
freeze-drying of the medium solution is not practicable, but exposure, holding times
in the freeze dryer should be as normal.
10.5 Semi-solid Products (e.g. Sterile Ointments and Creams): Simulate the normal
process cycle as closely as possible, filling a sterile liquid growth medium made to
similar consistency as the normal product by the addition, for example, of agar
(approximately 4 g. per litre) or carboxymethylcellulose.
11. REVALIDATION
11.1 Following initial aseptic process validation, media-fills and process simulations
should be repeated to an extent, and at a frequency, which will depend on the
occurrence of events or changes which may bear upon the potential microbial hazard
to the process and product. Significant modifications to equipment or facilities,
changes in personnel, undesirable trends in environmental monitoring results, and
sterility test failures may all indicate an immediate need to implement a full process
validation protocol (i.e. minimum of 3 consecutive successful media-fill runs) with
the facility in question taken out of service until any problems have been resolved,
and the results of the three media-fills have been evaluated and found acceptable.
11.2 In the absence of any significant changes, or of any other events giving cause for
concern, then a minimum re-test frequency should be twice per year per operator
shift or team, for each process line. For single shift operations, the minimum
frequency should be 3 times for each process line per year.
12. DOCUMENTATION
The following information should be prepared in summary form for the purposes of inspection and
evaluation by the appropriate authorities.
12.1 Overview
A comprehensive outline of the protocol followed in the validation of the process

should be prepared. The overview should indicate the steps performed, in proper
sequence, and should encompass:
a) the approach taken;
May 2003 20
b) justification of the approach based on the product factors;

c) summation of any modifications to the equipment required;
and
d) any modifications to the protocol resulting from the study.
12.2 Prevalidation
12.2.1 A full description of the aseptic fill equipment and ancillary systems and
report(s) confirming successful installation in accordance with the
Installation Qualification Procedures and certifying that the equipment and
systems, as installed, will perform consistently within defined limits.
12.2.2 Statement of the Environmental Standards designated for each stage of the
manufacturing process and certification of the conformity of any controlled
environment with the designated standard(s) during the studies (see Section
7).
12.3 Process Qualification
12.3.1 A summary of the procedures and controls for the following, as applied
routinely and during the validation studies:
S dispensing ingredients
S water quality and supply
S cleaning/disinfection/sterilization (as
appropriate) of all equipment, surfaces and
services
S sterilization of equipment, vessels and
pipelines
S filter integrity testing
S equipment set-up, start-up and adjustment
S clothing and gowning of personnel
12.3.2 Full Process Qualification Report, including:
S medium used
S volume filled
S number of units filled
S number of leakers rejected
S number of units incubated
S incubation temperature
S incubation time
S control organisms used
S filter integrity test results
S record of all in-process monitoring and
control results
May 2003 21
S summary of number and qualifications of

personnel involved in the studies
S policy and records relating to permitted
operator interventions (see 4.11)
S written procedures for all laboratory tests and
formally recorded results of all laboratory
tests, with an evaluation of those results
against criteria established in the study
protocol(s).
12.3.3 If retrospective validation was conducted, the details of the lot analysis and
process condition evaluation, including results of in-process controls, should
be compiled for the time period being assessed. Evidence of the equivalence
of the manufacturing conditions used for these lots to the current process
conditions, including calibration and maintenance history, is required.
Evidence that process/product failures and discrepancies were included in the
evaluation should be available.
12.4 Expert Evaluation
An evaluation of the entire study against the protocol requirements as outlined above
should be prepared and the conclusions drawn at each stage stated. The final
conclusions should reflect whether the protocol requirements were met.
The evaluation should include an assessment of the ability of the planned calibration
and maintenance programs for the equipment and instrumentation to maintain the
validated conditions (see Sections 6, 7 and 8). In addition, all process monitoring
and control procedures required to routinely ensure that the validated conditions are
maintained should be reported.
The evaluation should be signed by duly authorized officers of the organization who
were members of the team establishing the protocol, and who have appropriate
expertise in the area assigned to. Overall approval of the study should be authorized
by the head of the validation team and the head of the Quality Control Department.
May 2003 22
GMP Committee members

Name Title / Bureau Location
France Dansereau, Chair Manager, Inspection Unit, HPFBI* Ottawa, ON
Kim Dayman-Rutkus A/Manager, Policy and Regulations, HPFBI Ottawa, ON
Richard Ferland MRA Officer, HPFBI Longueuil, Que.
Francisco Fernandes Compliance Specialist Scarborough, Ont.
Taras Gedz Bureau of Pharmaceutical Sciences Ottawa, ON
Denis Girard Veterinary Drug Directorate Ottawa, ON
Raymond Giroux Drug Specialist, HPFBI Longueuil, Que.
Alicja Kasina Drug Specialist, Halifax, NS
Daryl Krepps Senior Regulatory Advisor, BGTD** Ottawa, Ont.
Stephen McCaul MRA Officer, HPFBI Scarborough, Ont.
Médic Ndayishimiye, Secretary Compliance Officer, HPFBI Ottawa, Ont.
Randy Stephanchew Drug Specialist, HPFBI Winnipeg, Man.
Stephane Taillefer Compliance Specialist, HPFBI Longueuil, Que.
Sheila Welock Drug Specialist, HPFBI Burnaby, BC
* Health Products and Food Branch Inspectorate

** Biologics and Genetic Therapies Directorate
May 2003 23

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Health Products and Food

May 30, 2003

I am pleased to inform you that a revised version of Guide 0006

Inquiries about this document can be addressed to Ms. France

Health Products and Food Branch Inspectorate

Ce document est aussi disponible en français.

3. Protocol Development and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

5. Data Review and Study Certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

7. Environmental Considerations: Clean Room Standards, Qualification and Monitoring . . 9

8. Equipment Qualification and Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

9. Media Fill Studies (Solution Products) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

10. Media Fill Studies (Non-Solution Products) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

GMP Committee members . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

The steps recommended in this guideline may be summarized as follows:

S As a pre-requisite, all studies should be conducted in accordance with a detailed, pre-

S Suitable CLEAN ROOM FACILITIES should be available, in terms both of the

S All processing equipment should be properly INSTALLED, QUALIFIED and

S The process should be REVALIDATED at intervals (See Section 11).

S Comprehensive DOCUMENTATION should be available to define, support and

2.1 In the context of this guide, Process Validation is defined as:

The action taken to demonstrate, and to provide documented evidence that

2.3.1 Installation Qualification, that is demonstrating and certifying that a

2.3.2 Operational Qualification, consists of demonstrating that the

2.4 None of these various phases need to be considered as entirely "water-tight"

Specification Equipment Qualification Process

2.5.1 Prospective Validation applies to new processes and new equipment,

2.5.2 Concurrent Validation applies to existing processes and equipment.

2.5.3 Retrospective Validation applies to existing processes and equipment,

S the overall process is understood

3. PROTOCOL DEVELOPMENT AND CONTROL

3.4 Protocols should specify the following in detail:

3.4.3 Installation and qualification requirements for new equipment.

3.4.5 Detailed, step-wise statement of actions to be taken in performing the

3.4.6 Assignment of responsibility for performing the study.

3.4.7 Statements on all test methodology to be employed, with a precise statement

3.4.8 Test equipment calibration requirements.

3.4.9 References to any relevant Standard Operating Procedures (SOPs).

4.3 Engineering/maintenance personnel should be fully trained and competent in the

4.10 As in routine production, Clean Room operators involved in validation studies

5. DATA REVIEW AND STUDY CERTIFICATION

5.1.1 These evaluations should be made as the information becomes available.

6.1 All laboratory tests (including physical, chemical and microbiological

6.4 All measuring/recording/indicating instruments employed in the studies should be

7. ENVIRONMENTAL CONSIDERATIONS: CLEAN ROOM STANDARDS,

S Room air filter integrity tests.

7.5 Room pressure differentials should be monitored on a continuous, on-going,

7.9 As appropriate to the type of manufacturing process, consideration needs to be given

S Bioburden check on bulk solution, prior to sterile filtration.

S Exposure of "Settle Plates" (Petri dishes of nutrient agar) at

S Use of Air Sampling devices to determine the number of

S Use of Contact Plates, or Swabs, to check the microbiological

8. EQUIPMENT QUALIFICATION AND MAINTENANCE

S Confirmed as having been constructed as specified.

S Properly installed and provided with all necessary functioning

S Confirmed as capable of operating consistently, within pre-

9. MEDIA FILL STUDIES (SOLUTION PRODUCTS)

Figure 1: Process Flow Diagram of Liquid Media Filling of vials

Vials Stoppers Overseals Dispense Medium Powder

Wash Wash Wash Prepare Solution

Sterilize Sterilize Sterilize Sterilize Liquid Medium

Fill / Stopper / Seal Aseptically

Inoculate Control Vials

Clarity: As "made-up", it should be clear, so as to allow for